Summary of Work: The complex and poorly understood process of lineage progression from neural stem cells to neuronal and glial phenotypes has come under increasing study using a variety of in vitro strategies. An important issue to be resolved is how neural stem cells are regulated to either self-renew or differentiate. In vivo these cells line the ventricles of the developing central nervous system (CNS) with variable numbers radiating processes to the pial surface (radial glial form of neural stem cell). The cells integrate signals derived from both carrier proteins in the cerebrospinal fluid filling the ventricles and other cells in the neuroepithelium lining the ventricles. Signals conveyed by carrier proteins and via cell-cell interactions, which could serve to regulate cell lineage progression, have not been eluciated. We have developed a novel strategy to isolate identified subpopulations of neural stem cells and differentiaing progenitors from the CNS during neurogenesis and gliogenesis. The strategy involves labeling of live cells with reagents identifying surface gangliosides, which are conserved throughout vertebrate evolution, and epitopes characteristic of cells undergoing apoptosis in conjunction with fluorescence-activated cell sorting (FACS). This FACS strategy permits prospective cellular and molecular studies of neural stem cells for the first time. During FY 2002 we focused primarily on eluciating the role of basic fibroblast growth factor (bFGF) in neural stem cell biology. Subpopulations of neural stem cells express bFGF and one of its principal receptors and bFGF is necessary and sufficient to sustain the self-renewal of neural stem cells without differentiation. Withdrawal of bFGF from defined, serum-free medium halted self-renewal and resulted in lineage progression primarily into an astrocyte lineage. Inclusion of EGF with bFGF led to self-renewal and differentiation of neurons and glial phenotypes. Imaging of Ca2+ levels in self-renewing and differentiating cells revealed bFGF-mediated regulation via pathways triggering release from intracellular stores and entry from extracellular sources. The characteristics of bFGF-mediated Ca2+ signaling were developmentally regulated and in the most differentiated phenotypes could no longer be detected with the well-established protocol. Thus, bFGF-mediated Ca2+ signaling is widespread and readily detectable among neural stem cells in self-renewal and undergoing lineage progression. We have used pharmacology to identify two lipid pathways transducing bFGF interactions with its receptor into Ca2+ signals. Pharmacological block of these pathways attenuates proliferation of neural stem cells and induces apoptosis. Similar results were obtained with a specific inhibitor of the tyrosine auto-phosphorylation reaction initiated by bFGF following interaction with its receptor. The same inhibitor affected baseline Ca2+ levels, indicating that bFGF-mediated autophosphorylation of neural stem cells regulates Ca2+ levels, which are critical for self-renewal and differentiation. Finally, we have carried out large-scale sorting of the neural stem cell and neuronal and glial populations emerging during the embryonic development of the cortex. cDNA microarray analysis of these populations in collaboration with Dr. Lynn Hudson's lab will be carried out to reveal gene clusters whose abundance correlates with different stages of cell lineage progression.